Hi,

my first question would be, what sample did you give to MS, the raw sample before SDS PAGE, the band cut from the gel or maybe electroelution of your sample from gel? I was told by our MS specialists, that working with SDS gels doen't work that well, except for tryptic digestions etc..

Did you try dialysis with very low MWCO membrane (depending of peptide mass) against water/low salt buffer and subsequent freeze-drying? I am working with an acidic protein (pI ~ 4) and it needs no salts etc. at right pH and can be freeze-dried without complications. After that, the peptide should be visible by mass.

Additionally, was the mass completely empty? Acidic peptides/proteins can contains several sodium atoms, sometimes even after desalting!

Hey Robert,

Thanks for your response.

I gave mass the raw sample, also band cut from the gel. The peptide size expected aroubd 1 kDa as per SDS PAGE, so it was not given tryptic digestion treatment as suggested not needed for such small molecule. It pI is around 3.

I concentrated the sample using reverse phase chromatography using solvent which required in MS.

Yes my MS technicial said there is nothing he can see. so i used sample desalted as well.

What else i can try. you suggestion might be helpful.

Hi,

reading this, I wonder why nothing can be seen in MS. Was it an ESI-MS or MALDI-MS, since I have no clue what a peptide that highly charged (pI~3) would do in ESI-MS (but that's only an idea, not based on knowledge).

Secondly, when it runs at ~1kDa, the mass may not even be ~1kDa. My protein (pI ~4) runs at ~33kDa on an SDS-Page with a mass of 20kDa, but it has almost no hydrophobic residues. So I guess it depends on the charge/hydrophobicity ratio of the peptide. Did your MS technical then focus on the right region? Did he screen different regions?

Hope I could help!

Hey,

thanks a lo tfor your information. Honestly i dont think technician look on different region. as i am not expert of MS so i didnt argue on it. MS use in my sample was ESI-MS. Do you think MALDI will be more helpful. and yes my peptide is a hydrophobic one. Can i have some more suggestion on it to get some more clue. !

Hi,

i am no MS expert either, but I know there are two types of ESI (+) and (-), since you can charge the droplet both ways. That may be a reason. MALDI is something worth checking.

BTW, did you do a typical SDS-PAGE or did you make a peptide/Schägger gel to determine the mass more precisely?

I did the peptide/schägger gel which made specially for peptides.

If it is a single protein you can do SDS-PAGE cut it and send for MALDI-TOF if Multiple proteins DO IEF folloerd by spot Extraction and MALDI-TOF or other peptide sequencing you can identify it 99%.

Thomas mahesh thanks a lot for your response. my peptide is single one and its clear visible in SDS PAGE. sample was done through ESI MS but no result as u can see in my last comment. So what could be the reason ? can you suggest. Do you think MALDI will sort out the problem. ? Your suggestion is warm welcomed...